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Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Fang B, Niu J, Ren H, Guo Y, Wang S - PLoS ONE (2014)

Bottom Line: By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation.The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively.A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China.

ABSTRACT
Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

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The influence of glycerol concentration.a. The influence of glycerol concentration for GDH; b. The influence of glycerol concentration for Mn-GDH. Reaction conditions: Glycerol concentration (×: 0.010 mol/L, 0.015 mol/L, Δ: 0.025 mol/L, O: 0.100 mol/L, □: 0.200 mol/L); enzyme, 1 mg/L; pH 12.0; temperature, 45°C.
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pone-0099162-g001: The influence of glycerol concentration.a. The influence of glycerol concentration for GDH; b. The influence of glycerol concentration for Mn-GDH. Reaction conditions: Glycerol concentration (×: 0.010 mol/L, 0.015 mol/L, Δ: 0.025 mol/L, O: 0.100 mol/L, □: 0.200 mol/L); enzyme, 1 mg/L; pH 12.0; temperature, 45°C.

Mentions: The influence of substrate concentration of both substrates, NAD+ and glycerol, on GDH (Fig. 1a) and Mn-GDH (Fig. 1b) were studied. Double reciprocal plots of six NAD+ concentrations versus reaction rates at six fixed glycerol concentrations were drawn [22]. Earlier studies that were conducted on glycerol dehydrogenase from Klebsiella pneumonia[23] and from other microorganisms [24], [25] reported the GDHs follow an ordered Bi-Bi sequential mechanism. Therefore, it was reasonable to assume that GDH from Klebsiella pneumonia and Mn-GDH obey the ordered Bi-Bi sequential mechanism. Kinetic parameters were determined from Lineweaver-Burke plots. The kinetic parameters for GDH and Mn-GDH are listed in Table 1.


Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Fang B, Niu J, Ren H, Guo Y, Wang S - PLoS ONE (2014)

The influence of glycerol concentration.a. The influence of glycerol concentration for GDH; b. The influence of glycerol concentration for Mn-GDH. Reaction conditions: Glycerol concentration (×: 0.010 mol/L, 0.015 mol/L, Δ: 0.025 mol/L, O: 0.100 mol/L, □: 0.200 mol/L); enzyme, 1 mg/L; pH 12.0; temperature, 45°C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4045801&req=5

pone-0099162-g001: The influence of glycerol concentration.a. The influence of glycerol concentration for GDH; b. The influence of glycerol concentration for Mn-GDH. Reaction conditions: Glycerol concentration (×: 0.010 mol/L, 0.015 mol/L, Δ: 0.025 mol/L, O: 0.100 mol/L, □: 0.200 mol/L); enzyme, 1 mg/L; pH 12.0; temperature, 45°C.
Mentions: The influence of substrate concentration of both substrates, NAD+ and glycerol, on GDH (Fig. 1a) and Mn-GDH (Fig. 1b) were studied. Double reciprocal plots of six NAD+ concentrations versus reaction rates at six fixed glycerol concentrations were drawn [22]. Earlier studies that were conducted on glycerol dehydrogenase from Klebsiella pneumonia[23] and from other microorganisms [24], [25] reported the GDHs follow an ordered Bi-Bi sequential mechanism. Therefore, it was reasonable to assume that GDH from Klebsiella pneumonia and Mn-GDH obey the ordered Bi-Bi sequential mechanism. Kinetic parameters were determined from Lineweaver-Burke plots. The kinetic parameters for GDH and Mn-GDH are listed in Table 1.

Bottom Line: By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation.The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively.A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China.

ABSTRACT
Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Show MeSH
Related in: MedlinePlus